Covalent modifications of protein by phosphorylation and oxidation are important mechanisms for the modulation of numerous cellular functions. Protein phosphorylation by protein kinase C (PKC) has been linked to the regulation of growth, differentiation, secretion, cellular metabolism, memory, and sensory transduction. Multiple PKC subspecies have been identified by molecular cloning and the various isozymes have been shown to have distinct tissue, cellular, and subcellular distributions and are differentially expressed during development. The functional roles of these kinases in the control of cellular differentiation were tested with N1E115 neuroblastoma cells. PKC-activating phorbol ester counteracts the cAMP-induced neurite outgrowth of these cells by selective translocation and downregulation of PKC alpha and delta, whereas PKC beta and zeta are not greatly affected. PMA stimulates phosphorylation of several proteins including a 95 kDa elongation factor 2, its phosphorylation is attenuated by cAMP. Elucidation of the PKC-regulated events also places emphasis in the characterization of the substrate. A novel 28 kDa PKC/casein kinase II substrate has been identified and its cDNA cloned. This protein is a substrate of casein kinase II in N1E115 neuroblastoma cells. The extent of phosphorylation and protein level are cell cycle-regulated. Another potential PKC substrate, p14.4, has been identified and its cDNA cloned. This protein can also be phosphorylated by p34cdc2 kinase. Neurogranin (Ng) is a calmodulin (CaM)-binding PKC substrate. Its phosphorylation by PKC reduces the affinity for CaM and thus frees CaM for the CaM- dependent enzymes. In addition, oxidation of Ng in air or by peroxide or nitric oxide results in intramolecular disulfide bond formation and reduces the affinity of this protein for CaM. Site-directed mutagenesis of the four Cys residues in Ng has lead to identification of a pair of Cys residues involved in disulfide bond formation. Ng is an excellent acceptor for nitric oxide; the rate of nitrosation of Ng sulfhydryl groups and subsequent disulfide bond formation is even faster than one of the best known reductants, DTT. Modification of Ng by oxidation could be an important signaling mechanism of nitric oxide in the CNS. The genomic structures of the CNS-specific PKC gama and Ng were analyzed for the purpose of defining tissue-specific and development-regulated expression of these two genes. The promoter activity of the Ng gene can be stimulated by phorbol ester or by co-transfection with PKC cDNAs. A 20 kDa protein that binds to the AT-rich regions of the PKC gamma and Ng gene promoters has been identified; phosphorylation of this protein by PKC attenuates its binding to DNA elements containing ATTA, ATAA and AATA motifs.